A Preliminary Study On The Antitumor Effect And Immune Effect Of IRE Combined With Immune Adjuvant In Pancreatic Cancer

ObjectiveTo study the anti-tumor and immunomodulatory effects of irreversible electroablation technology combined with immune adjuvant GC on mouse pancreatic cancer pan02,and to explore the death mode of mouse pancreatic cancer pan02 and whether it is related to mitochondrial apoptosis.To provide experimental basis for optimizing the treatment mode of irreversible electrolysis,improving the microenvironment of mouse pancreatic cancer and improving the efficacy of electroablation.Methods1.Antitumor effect of irreversible electroablation combined with GC on pancreatic cancer in mice: In animal experiments,5~6-week-old female c57bl/6mice were prepared with skin,and 1×106 pancreatic cancer cells were inoculated subcutaneously on the right back to construct a subcutaneous transplantation tumor model,and when the longest diameter of implant tumors reached about6~7mm,tumor-bearing mice were randomly divided into control group,irreversible electroablation group and irreversible electroablation combined with GC group,with10 in each group.The control group was not treated,the IRE treatment group used a field strength of 1000 v/cm,pulse 60 times/min to treat the tumor,and the combined group was treated with an IRE voltage of 1000v/cm and pulse 60times/min,and injected 100μL of immune adjuvant GC,and the survival rate of mice was observed every 2 days after treatment,and the anti-tumor effects of different treatment options were compared.In vitro cell experiments,when subculturing pancreatic cancer cells of PAN-02,the coverslips were first autoclaved and put into a petri dish,the cells were seeded into the dish to grow on the slide,and after the cells were close to growing into a monolayer,they were randomly divided into a control group,an irreversible electrochloricidal treatment group,and 3Petri dishes per group.The control group was treated without treatment,and the IRE group was treated with a field strength of 700 v/cm,pulse 60 times/min,and after treatment,FITC-GC solution was added to each Petri dish and incubated for about 3 hours,and coverslips were taken out for observation under a confocal microscope.2.Immunomodulatory effect of irreversible electroablation combined with GC on pancreatic cancer in mice: repeat the above steps to construct a subcutaneous tumor model of pancreatic cancer in mice,and when the longest diameter of the tumor reaches about 6~7mm,different groups of mouse tumors are treated according to the above experimental protocol,3~5 mice in each group.On the 10 th day after IRE treatment,the transplanted tumors and lymph nodes of mice were taken:(1)Western Blot was used to detect heat shock protein-70 and high-mobility group protein-1 in tumor tissues,and the effects of different treatments on damage-related molecular patterns were observed;(2)Flow cytometry and immunohistochemical staining were used to detect CD4+ T cells,CD8+ T cells,Treg cells,memory T cells and effector T cells in tumor tissues and lymph nodes,and the effects of different treatment groups on the number of infiltrated immune cells were observed;(3)Flow cytometry was used to detect the expression of PD-1and LAG-3 on the surface of CD4+ T cells and CD8+ T cells in tumor tissues and lymph nodes.Results1.Anti-tumor effect of irreversible electroporation combined with GC on mouse pancreatic cancer: In vitro experiments,IRE combined with GC can enhance the killing effect on pancreatic cancer cells and reduce the growth rate of pancreatic cancer cells;When the pulse was fixed,the stronger the field intensity was,the lower the survival rate of tumor cells was.When the field intensity is the same,the survival rate will gradually decrease with more pulses.And in animal studies,the combination therapy extended the survival time of tumor bearing mice.It was shown that adectin V can block preferential binding of GC to IRE treated cells.This protein has a high propensity to bind to acidic phosphatidylserine and has a particularly strong affinity for phosphatidylserine,suggesting that phosphatidylserine may be a key site for GC binding.It is speculated that GC interaction with IRE may lead to increased cell death by hindering the repair of affected membrane structures.2.The immunoregulatory effect of irreversible electroporation combined with GC on pancreatic cancer in mice: compared with the control group,the experimental group could increase the number of CD4+ T cells and CD8+ T cells in tumor tissues to varying degrees,increase the number of large blood vessels in tumor tissues,and reduce the expression of PD-1 and LAG-3 in tumor tissues.The tumor growth index,fibroblast and TREG cell numbers were also decreased,and the combined treatment effect was more significant.In addition,the number of memory T cells in lymph nodes and effector T cells in tumor tissues was increased in the combined treatment group.ConclusionsUnder the electrical parameters in this study,irreversible electroporation can obviously kill tumor cells,and the combination of GC can produce synergistic therapeutic effect.The results showed that irreversible electroporation combined with GC up-regulated the number of T lymphocytes,reduced the number of immunosuppressive cells,enhanced the systemic anti-tumor immunity,and reduced Ki-67 and FAP-α to reshape the tumor microenvironment.In addition,combined therapy reduced mitochondrial membrane potential and GC was speculated to promote tumor cell death through membrane structure repair.